Biomedical subjects

Raymond E Meyn

Publications and source records attributed to Raymond E Meyn.

At least 19 recordsLinked to original sources

PubMed2006

Targeted apoptosis activation with GrB/scFvMEL modulates melanoma growth, metastatic spread, chemosensitivity, and radiosensitivity.

GrB/scFvMEL, a fusion protein composed of human granzyme B (GrB) and the single-chain antibody scFvMEL, targets melanoma gp240 antigen and exerts impressive cytotoxic effects by inducing apoptosis. We evaluated the effects of GrB/scFvMEL on chemotherapy, radiation therapy, metastasis in vitro, and the growth of human melanoma A375 xenograft tumors in nude mice. GrB/scFvMEL showed synergistic cytotoxicity when coadministered with doxorubicin, vincristine or cisplatin, and additive effects, in combination with etoposide or cytarabine. Optimal cytotoxic effects were obtained when cells were treated first with GrB/scFvMEL followed by exposure to the agent (rather than the reverse). Pretreatment of A375 cells with GrB/scFvMEL significantly sensitized melanoma cells to ionizing radiation assessed using a clonogenic survival assay. Subtoxic doses of GrB/scFvMEL inhibited the invasion of A375 cells into Matrigel. GrB/scFvMEL (37.5 mg/kg) was administered intravenously to nude mice bearing A375 tumors. Saline-treated tumors increased 24-fold, whereas tumors treated with GrB/scFvMEL showed a significant tumor growth delay increasing four-fold. Tumor tissue displayed an increase in apoptotic nuclei compared to control. Thus, the targeted delivery of GrB to tumors may have a significant potential for cancer treatment. Targeted therapeutic agents specifically designed to impact cellular apoptotic pathways may represent a novel class of therapeutic agents.

Animals↗

PubMed2006

MDA-7/IL-24-based cancer gene therapy: translation from the laboratory to the clinic.

Despite recent advances in treatment strategies, the overall 5-year survival rate for patients with common epithelial cancers is poor largely because of the difficulty in treating metastatic cancers. Therefore, therapeutic agents are urgently needed that can effectively inhibit both primary epithelial tumors and their metastases. One such agent that has shown promise in preclinical studies is the tumor suppressor/cytokine, melanoma differentiation associated gene-7 also known as interleukin-24 (mda-7/IL-24). Preclinical studies from our and other laboratories have shown that overexpression of MDA-7/IL-24 causes a strong tumor- suppressive effect in many human cancer cells but spares normal cells. This gene therapy also enhances the tumor-suppressive activity of radiotherapy and chemotherapy. Secreted MDA-7 protein that is glycosylated also has been shown to have potent antiangiogenic activity both in vitro and in vivo. Studies examining the immune properties of mda-7 have shown that MDA-7/IL-24 unlike the related IL-10, functions as a Th1 cytokine. Recently, an MDA-7 protein-mediated "bystander effect" on tumor cells has been documented. Building on these findings we successfully completed a Phase I clinical trial of adenovirus-based mda-7 cancer therapy that confirmed the safety of this gene therapy. Phase II trials evaluating the efficacy of mda-7-based gene therapy are warranted. The outcome of such ongoing mda-7-based gene therapy trials will allow us to better understand this therapy's clinical utility.

Adjuvants, Immunologic↗

PubMed2005

Histone deacetylase inhibitors radiosensitize human melanoma cells by suppressing DNA repair activity.

PURPOSE: Histone deacetylase (HDAC) inhibitors have emerged recently as promising anticancer agents. They arrest cells in the cell cycle and induce differentiation and cell death. The antitumor activity of HDAC inhibitors has been linked to their ability to induce gene expression through acetylation of histone and nonhistone proteins. However, it has recently been suggested that HDAC inhibitors may also enhance the activity of other cancer therapeutics, including radiotherapy. The purpose of this study was to evaluate the ability of HDAC inhibitors to radiosensitize human melanoma cells in vitro. EXPERIMENTAL DESIGN: A panel of HDAC inhibitors that included sodium butyrate (NaB), phenylbutyrate, tributyrin, and trichostatin A were tested for their ability to radiosensitize two human melanoma cell lines (A375 and MeWo) using clonogenic cell survival assays. Apoptosis and DNA repair were measured by standard assays. RESULTS: NaB induced hyperacetylation of histone H4 in the two melanoma cell lines and the normal human fibroblasts. NaB radiosensitized both the A375 and MeWo melanoma cell lines, substantially reducing the surviving fraction at 2 Gy (SF2), whereas it had no effect on the normal human fibroblasts. The other HDAC inhibitors, phenylbutyrate, tributyrin, and trichostatin A had significant radiosensitizing effects on both melanoma cell lines tested. NaB modestly enhanced radiation-induced apoptosis that did not correlate with survival but did correlate with functional impairment of DNA repair as determined based on the host cell reactivation assay. Moreover, NaB significantly reduced the expression of the repair-related genes Ku70 and Ku86 and DNA-dependent protein kinase catalytic subunit in melanoma cells at the protein and mRNA levels. Normal human fibroblasts showed no change in DNA repair capacity or levels of DNA repair proteins following NaB treatment. We also examined gamma-H2AX phosphorylation as a marker of radiation response to NaB and observed that compared with controls, gamma-H2AX foci persisted long after ionizing exposure in the NaB-treated cells. CONCLUSIONS: HDAC inhibitors radiosensitize human tumor cells by affecting their ability to repair the DNA damage induced by ionizing radiation and that gamma-H2AX phosphorylation can be used as a predictive marker of radioresponse.

Acetylation↗

PubMed2005

Clonogenic cell survival assay.

The clonogenic cell survival assay determines the ability of a cell to proliferate indefinitely, thereby retaining its reproductive ability to form a large colony or a clone. This cell is then said to be clonogenic. A cell survival curve is therefore defined as a relationship between the dose of the agent used to produce an insult and the fraction of cells retaining their ability to reproduce. Although clonogenic cell survival assays were initially described for studying the effects of radiation on cells and have played an essential role in radiobiology, they are now widely used to examine the effects of agents with potential applications in the clinic. These include, in addition to ionizing radiation, chemotherapy agents such as etoposide and cisplatin, antiangiogenic agents such as endostatin and angiostatin, and cytokines and their receptors, either alone or in combination therapy. Survival curves have been generated for many established cell lines growing in culture. One can use cell lines from various origins including humans and rodents; these cells can be neoplastic or normal. Because survival curves have wide application in evaluating the reproductive integrity of different cells, we provide here the steps involved in setting up a typical experiment using an established cell line in culture.

Animals↗

PubMed2004

Adenoviral-mediated mda-7 expression suppresses DNA repair capacity and radiosensitizes non-small-cell lung cancer cells.

The melanoma differentiation-associated gene-7 (mda-7) was identified by virtue of its enhanced expression in human melanoma cells induced into terminal differentiation. Enforced expression of mda-7 in human cancer cell lines of diverse origins results in the suppression of growth and induction of apoptosis. We have shown that adenoviral-mediated mda-7 (Ad-mda7) radiosensitizes non-small-cell lung cancer (NSCLC) cells by enhancing the apoptotic pathway. To identify the mechanism of this radiosensitization, we examined the level of proteins involved in the nonhomologous end-joining (NHEJ) pathway of DNA double-strand break (DSB) repair. Western blot analysis indicated that the expression of NHEJ pathway components Ku70, XRCC4, and DNA ligase IV was downregulated in NSCLC cells--A549 with Ad-mda7 treatment. No such change was observed in normal human CCD16 fibroblasts previously shown not to be radiosensitized by Ad-mda7. The biological significance of these changes of expression of proteins critical for repair of radiation-induced DSBs was confirmed via the analysis of DSB rejoining kinetics using pulsed field gel electrophoresis and assessment of host cell reactivation capacity following Ad-mda7 treatment. Based on these results, we hypothesize that Ad-mda7 sensitizes NSCLC cells to ionizing radiation by suppressing the activity of NHEJ, a pathway essential for repair of radiation-induced DSBs.

Adenoviridae↗

PubMed2004

A comprehensive assessment of p53-responsive genes following adenoviral-p53 gene transfer in Bcl-2-expressing prostate cancer cells.

The p53 protein can induce cell cycle arrest or apoptosis following activation in response to DNA damage. The function of p53 is largely mediated by regulating the expression of downstream target genes. Adenoviral-p53 gene transfer (Ad-p53) is currently being evaluated in clinical trials as a therapeutic intervention. Tumor response is likely to be influenced by context-dependent variables, such as expression of bcl-2. Bcl-2 is upregulated in a variety of neoplasms, and can inhibit p53-dependent apoptosis. It was therefore of interest to use a global genomic strategy to assess gene expression following Ad-p53 gene transfer and to determine if the expression of specific Ad-p53-responsive genes could be modulated in the context of bcl-2 gene deregulation. cDNA arrays were used to identify p53-responsive genes following Ad-p53 gene transfer in control and bcl-2-overexpressing PC3 prostate cancer cells. A total of 40 transcripts were significantly upregulated by Ad-p53 in both control and bcl-2-transfectant PC3 cells. Conversely, 19 transcripts were significantly repressed in both cell lines. These Ad-p53-responsive transcripts included previously identified p53 targets, known genes representing candidate p53 targets, and transcripts identified as expressed sequence tags. A subset of 15 transcripts was differentially modulated by Ad-p53 in the context of bcl-2. Some of these genes were also differentially modulated in LNCaP (wt p53) cells following DNA damage. These results document a number of potential p53 targets and mediators of therapeutically relevant genotoxic stress. The findings further suggest that bcl-2 may inhibit cell death at multiple points downstream of p53 activation.

Adenoviridae↗

PubMed2004

Predicting radiosensitivity using DNA end-binding complex analysis.

Previous reports have suggested that measuring radiosensitivity of normal and tumor cells would have significant clinical relevance for the practice of radiation oncology. We hypothesized that radiosensitivity might be predicted by analyzing DNA end-binding complexes (DNA-EBCs), which form at DNA double-strand breaks, the most important cytotoxic lesion caused by radiation. To test this hypothesis, the DNA-EBC pattern of 21 primary human fibroblast cultures and 15 tumor cell lines were studied. DNA-EBC patterns were determined using a modified electrophoretic mobility shift assay and were correlated with radiosensitivity, as measured by SF2. DNA-EBC analysis identified a rapidly migrating ATM-containing band (identified as "band-A") of which the density correlated with SF2 (0.02 </= SF2 </= 0.41) in primary fibroblasts (r(2) = 0.77). The DNA-EBC pattern of peripheral blood lymphocytes was identical to that of fibroblasts. In addition, band-A density correlated with SF2 (0.35 </= SF2 </= 0.80) in 15 human tumor cell lines (r(2) = 0.91). Densitometry of other bands, or total DNA-EBC binding, correlated more poorly with SF2 (r(2) < 0.45). These data indicate that DNA-EBC analysis may be a practical, clinically relevant predictor of tumor and primary cell radiosensitivity.

Cell Line↗

PubMed2004

Adenovirus-mediated mda-7 (IL24) gene therapy suppresses angiogenesis and sensitizes NSCLC xenograft tumors to radiation.

Melanoma differentiation-associated gene-7 (mda-7), recently classified as interleukin-24 (approved gene symbol IL24), is thought to be a tumor suppressor gene based on the loss of its expression in many different types of cancer. Gene therapy by adenovirus-mediated mda-7 (Ad-mda7) gene transfer has been shown to inhibit the growth of several different tumor cell lines, in vitro and in vivo. We previously demonstrated that Ad-mda7 radiosensitized non-small-cell lung cancer (NSCLC) cell lines by enhancing an apoptosis pathway through the activation of JNK and c-Jun. In the present study, we investigated the efficacy of intratumoral administration of Ad-mda7 combined with ionizing radiation for treating A549 xenograft tumors in nude mice. Substantial and long-lasting inhibition of tumor growth was evident following the combined treatment. Histological examination revealed marked reduction of angiogenic factors (bFGF, VEGF) and microvessel density and enhanced apoptosis in the tumors treated with the combination therapy compared to those treated with Ad-mda7 alone or radiation alone. To confirm the radiosensitizing effect of secreted MDA-7 protein, we performed clonogenic survival assays using human umbilical vein endothelial cells (HUVECs), A549 cells, and normal human lung fibroblasts, CCD16 cells, pretreated with the conditioned medium from 293 cells that had been stably transfected with mda-7 or a control vector. The results showed that MDA-7 protein sensitized HUVECs to ionizing radiation but not A549 cells or CCD16 cells. Our results suggest that Ad-mda7 in combination with radiation enhances apoptosis in the tumors and that secreted MDA-7 protein inhibits angiogenesis by sensitizing endothelial cells to ionizing radiation without affecting other normal cells. We conclude that the combination of mda-7 gene therapy and radiotherapy may be a feasible and effective strategy for treatment of NSCLC.

Adenoviridae↗

PubMed2004

Adenoviral-mediated PTEN transgene expression sensitizes Bcl-2-expressing prostate cancer cells to radiation.

Bcl-2 is associated with resistance to radiotherapy in prostate cancer. It was recently demonstrated that transduction of LNCaP prostate cells with the PTEN gene resulted in Bcl-2 downregulation. We hypothesized that forced expression of PTEN in prostate cancer cells would sensitize cells to radiation, downregulate Bcl-2 expression, and potentiate the G2M block induced by radiation. Four cell lines - PC-3-Bcl-2 (Bcl-2 overexpression, deleted PTEN), PC-3-Neo (wild-type Bcl-2, deleted PTEN), LNCaP (Bcl-2 overexpression, deleted PTEN), and DU-145 (wild-type Bcl-2 and PTEN) - were transduced with a recombinant adenovirus-5 vector expressing the human wild-type PTEN cDNA under the control of a human cytomegalovirus promoter (Ad-MMAC). After correction for the effect of Ad-MMAC on plating efficiency, Ad-MMAC treatment reduced the surviving fractions after 2 Gy as follows: PC-3-Bcl-2, from 60.5 to 3.6%; PC-3-Neo, no reduction; LNCaP, from 29.6 to 16.3%; and DU-145, from 32.7 to 25.7%. PTEN expression was associated with the downregulation of Bcl-2 expression in PC-3-Bcl-2 and LNCaP cell lines. Ad-MMAC plus radiotherapy potentiated the G2M block seen with radiotherapy alone only in PC-3-Bcl-2 cells. These findings suggest that overexpression of Bcl-2 result in radioresistance and inability of radiation to cause its typical G2M cell-cycle arrest.

Adenoviridae↗

PubMed2004

GSH depletion enhances adenoviral bax-induced apoptosis in lung cancer cells.

The utility of dominant acting proapoptotic molecules to induce cell death in cancer cells is being evaluated in preclinical studies and clinical trials. We recently developed a binary adenoviral expression system to enable the efficient gene transfer of Bax and other proapoptotic molecules. Using this system, overexpression of Bax protein in four non-small-cell lung cancer (NSCLC) cell lines, H1299, A549, H226 and H322, was evaluated. The H322 line exhibited significant resistance to Bax-induced cell death compared to the other cell lines. H322 cells had the highest level of glutathione (GSH). GSH levels were significantly decreased following buthionine sulfoximine treatment and this coincided with enhanced apoptosis induction by Ad-Bax in H322 cells. GSH depletion enhanced Bax protein translocation to mitochondrial membranes. These findings suggest that the redox status may be a determinant of Bax-mediated cell death and that manipulation of intracellular thiols may sensitize cells to apoptosis by facilitating Bax insertion into mitochondrial membranes.

Adenoviridae↗

PubMed2004

Inhibition of constitutively activated nuclear factor-kappaB radiosensitizes human melanoma cells.

Melanoma tumors and cultured cell lines are relatively resistant to the cytotoxic effects of ionizing radiation, thereby limiting the use of radiotherapy for the clinical treatment of melanoma. New strategies for sensitizing melanoma cells therefore deserve examination. In an attempt to identify and target signaling pathways that contribute to radioresistance, we investigated the role of nuclear factor-kappaB (NF-kappaB), a transcription factor known to inhibit apoptosis induced by a variety of stimuli and promote radioresistance. Two human metastatic melanoma cell lines, A375 and MeWo, were used to examine the radiosensitizing effects of inhibitors of the NF-kappaB pathway. Nuclear extracts from these cell lines were tested for active NF-kappaB using the electrophoretic mobility shift assay. Both melanoma cell lines had constitutively activated NF-kappaB as observed by electrophoretic mobility shift assay. In an attempt to reverse NF-kappaB activity, cells were treated either with vehicle alone (DMSO) or with a proteasome inhibitor Z-Leu-Leu-Leu-H (MG132; 10 micromol/L for 2 hours prior to irradiation) that inhibited both constitutive and radiation-induced NF-kappaB activity. The clonogenic cell survival assay showed that pretreatment with MG132 enhanced tumor cell radiosensitivity with the survival factor at 2 Gy being reduced from 48 +/- 0.8% and 48 +/- 1.6% in vehicle-treated cells to 27.7 +/- 0.32% and 34.3 +/- 0.7% in MG132-treated MeWo and A375 cells, respectively. To test the role of NF-kappaB in radioresistance more directly, MeWo cells were stably transfected with a dominant-negative mutant IkappaBalpha construct, which led to the inhibition of both constitutive and radiation-induced NF-kappaB activity. A modest restoration of radiosensitivity was also observed in the stably transfected MeWo cells with survival factor at 2 Gy values being reduced from 47 +/- 0.8% in parental MeWo cells to 32.9 +/- 0.7% in stable transfectants. Because constitutively activated mitogen-activated protein kinase kinase (MEK) pathway has been shown to lead to activated NF-kappaB, we wanted to determine the relative contribution of activated MEK in the human melanoma cells. To test this, MeWo and A375 melanoma cells were exposed to the MEK inhibitor PD184352. Treatment with PD184352 partially reversed NF-kappaB activity but did not impart radiation sensitivity to these cells. Our results indicate that activated NF-kappaB may be one of the pathways responsible for the radioresistance of melanoma cells and that strategies for inhibiting its influence may be useful in restoring the radioresponse of melanomas.

Benzamides↗

PubMed2003

The MEK/ERK pathway acts upstream of NF kappa B1 (p50) homodimer activity and Bcl-2 expression in a murine B-cell lymphoma cell line. MEK inhibition restores radiation-induced apoptosis.

In a previously published report (Kurland, J. F., Kodym, R., Story, M. D., Spurgers, K. B., McDonnell, T. J., and Meyn, R. E. (2001) J. Biol. Chem. 276, 45380-45386), we described the NF kappa B status for two murine B-cell lymphoma cell lines, LY-as (apoptosis-sensitive) and LY-ar (apoptosis-refractory) and provided evidence that NF kappa B1 (p50) homodimers contribute to the expression of Bcl-2 in the LY-ar line. In the present study, we investigated the upstream signals leading to p50 homodimer activation and Bcl-2 expression. We found that in LY-ar cells, ERK1 and ERK2 were constitutively phosphorylated, whereas LY-as cells had no detectable ERK1 or ERK2 phosphorylation. Treatment of LY-ar cells with the MEK inhibitors PD 98059, U0126, and PD 184352 led to a loss of phosphorylated ERK1 and ERK2, a reversal of nuclear p50 homodimer DNA binding, and a decrease in Bcl-2 protein expression. Similarly, activation of the MEK/ERK pathway in LY-as cells by phorbol ester led to Bcl-2 expression that could be blocked by PD 98059. Furthermore, treatment of LY-ar cells with tumor necrosis factor-alpha, an I kappa B kinase activator, did not alter the suppressive effect of PD 98059 on p50 homodimer activity, suggesting an I kappa B kinase-independent pathway for p50 homodimer activation. Lastly, all three MEK inhibitors sensitized LY-ar cells to radiation-induced apoptosis. We conclude that the MEK/ERK pathway acts upstream of p50 homodimer activity and Bcl-2 expression in this B-cell lymphoma cell system and suggest that the use of MEK inhibitors could be useful clinically in combination with ionizing radiation to treat lymphoid malignancies.

Animals↗

PubMed2003

Translation inhibitors sensitize prostate cancer cells to apoptosis induced by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) by activating c-Jun N-terminal kinase.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in several human tumors both in vitro and in vivo, however, some tumors remain resistant for poorly understood reasons. Using a quantitative DNA fragmentation assay for apoptosis, we have shown that human prostate cancer cells are resistant to a wide range of TRAIL doses up to 500 ng/ml. However, translation inhibitors, such as anisomycin, cycloheximide, emetine, harringtonine, and puromycin, unlike several transcription inhibitors, significantly sensitized PC3-neomycin (PC3-neo) cells to TRAIL-induced apoptosis. These effects were inhibited in PC3 cells engineered to express bcl2 (PC3-bcl2). Translation inhibitors led to activation of c-Jun N-terminal kinase (JNK), which plays a role in this sensitization process because inhibition of JNK activation resulted in protection against TRAIL plus translation inhibitor-induced apoptosis. JNK activation may be required for this process, but it is not sufficient because activation of JNK using an MEKK2 expression vector did not mimic the sensitizing effect of translation inhibitors. Other stress-activated protein kinases, such as ERK and p38, play an insignificant role in determining the apoptotic sensitivity. We conclude that activation of JNK is required for sensitization of PC3 cells to TRAIL-induced apoptosis by translation inhibitors in cells that are otherwise TRAIL-resistant. However, in addition to JNK activation, other aspects of translation inhibition such as the suppressed activity of apoptosis-inhibitory proteins or activation of other signal transduction pathways must also be involved.

Activating Transcription Factor 2↗

PubMed2003

MDA-7 negatively regulates the beta-catenin and PI3K signaling pathways in breast and lung tumor cells.

mda-7 is a novel tumor suppressor with cytokine properties. Adenoviral mda-7 (Ad-mda7) induces apoptosis and cell death selectively in tumor cells. The molecular mechanisms underlying the anti-tumor activity of Ad-mda7 in breast and lung cancer lines were investigated. Microarray analyses implicated both the beta-catenin and the PI3K signaling pathways. Ad-mda7 treatment increased protein expression from tumor suppressor genes, including E-cadherin, APC, GSK-3beta, and PTEN, and decreased expression of proto-oncogenes involved in beta-catenin and PI3K signaling. Ad-mda7 caused a redistribution of cellular beta-catenin from the nucleus to the plasma membrane, resulting in reduced TCF/LEF transcriptional activity, and upregulated the E-cadherin-beta-catenin adhesion complex in a tumor cell-specific manner. Expression of the PI3K pathway members (p85 PI3K, FAK, ILK-1, Akt, and PLC-gamma) was downregulated and expression of the PI3K antagonist PTEN was increased. Consistent with this result, pharmacological inhibition of PI3K by wortmannin did not abrogate killing by Ad-mda7. Killing of breast cancer cells by Ad-mda7 required both MAPK and MEK1/2 signaling pathways, whereas these pathways were not essential for MDA-7-mediated killing in lung cancer cells. Thus, in breast and lung tumor cells MDA-7 protein expression modulates cell-cell adhesion and intracellular signaling via coordinate regulation of the beta-catenin and PI3K pathways.

Adenoviridae↗

PubMed2003

Report from the Radiation Oncology Committee of the Southwest Oncology Group (SWOG): Research Objectives Workshop 2003.

To achieve the ultimate goal of cancer treatment, which is 100% cancer control with negligible toxicity, the therapeutic window must be enlarged, allowing for higher doses of beneficial treatments with reduced toxicity. The advent of image- and metabolism-guided therapy offers the best opportunity to date for combining modern radiation targeting and imaging techniques. Indeed, for the first time, it is reasonable to locally target metastatic disease with the goal of sterilization. Combining these focal radiation techniques with novel targeted antiproliferative agents and full-dose classic cytotoxic chemotherapy will become more effective as we learn to use these compounds in a less systemically toxic manner and as radiation fields become more defined. In addition, increasing numbers of biologic modifiers of normal tissue response are becoming available, and they suggest great promise for decreasing the normal tissue toxicity resulting from both radiation and chemotherapy treatments. Thus, radiation metastectomy for gross metastases, used together with systemic control of micrometastatic disease, may yield improved survival rates. This hypothesis is ready for testing in cancers of the breast, prostate, colon, and in sarcomas. Enlarging the therapeutic window is a major goal that would allow for an increasingly favorable therapeutic gain.

Antineoplastic Agents↗

PubMed2003

Adenovirus-mediated wild-type p53 radiosensitizes human tumor cells by suppressing DNA repair capacity.

Functional inactivation of the p53 gene and robust DNA repair capacity may be among the salient causes of radioresistance in tumor cells. We expressed the wild-type (wt) p53 gene in a p53-mutant human epidermoid carcinoma cell line, A431, using an adenoviral vector [adenovirus-p53 (Ad-p53), INGN 201], examined its radiosensitivity, and correlated p53 status and radiosensitivity with cellular repair functions. Using clonogenic survival assays and the terminal deoxynucleotidyl transferase-mediated nick end labeling assay for apoptosis, we demonstrated that preirradiation treatment with Ad-p53 significantly increased the radiosensitivity of A431 cells over controls. Induction of p53 expression using a construct where p53 expression was under the control of an inducible promoter also significantly increased radiosensitivity of H1299 lung tumor cells, which are otherwise null for p53. These results did not correlate with radiation-induced apoptosis but did correlate with functional impairment of DNA repair and suppressed expression of several repair-related genes, such as Ku70, DNA-dependent protein kinase, ataxia telangiectasia mutated, and X-ray-sensitive complementation group 4. Normal human fibroblast MRC-9 cells showed no impairment in the repair capability due to Ad-p53 despite the suppression of some repair genes. Expression of Ku70, which is known to mediate diverse cellular functions, correlated with the differential effects of p53 on radiosensitivity in the normal and tumor cells.

Adenoviridae↗

PubMed2002

Inhibition of human lung cancer growth following adenovirus-mediated mda-7 gene expression in vivo.

Overexpression of the melanoma differentiation associated gene-7 (mda-7) in vitro results in suppression of lung cancer cell proliferation. However, the ability of MDA-7 to suppress lung cancer in vivo has not been previously demonstrated. In this study, we investigated the possibility of inducing overexpression of the mda-7 gene in human non-small cell lung carcinoma cells in vivo and its effects on tumor growth. Adenovirus-mediated overexpression of MDA-7 in p53-wild-type A549 and p53-null H1299 subcutaneous tumors resulted in significant tumor growth inhibition through induction of apoptosis. In addition, decreased CD31/PECAM expression and upregulation of APO2/TRAIL were observed in tumors expressing MDA-7. In vivo studies correlated well with in vitro inhibition of lung tumor cell proliferation and endothelial cell differentiation mediated by Ad-mda7. These data demonstrate that Ad-mda7 functions as a multi-modality anti-cancer agent, possessing both, pro-apoptotic and anti-angiogenic properties. We demonstrate for the first time the potential therapeutic effects of Ad-mda7 in human lung cancer.

Adenoviridae↗

PubMed2002

Adenovirus-mediated mda-7 gene expression radiosensitizes non-small cell lung cancer cells via TP53-independent mechanisms.

We examined the ability of adenoviral-mediated expression of the melanoma differentiation associated gene-7 (Ad-mda-7), to radiosensitize non-small cell lung cancer (NSCLC) cell lines (A549 (wt-TP53/wt-RB1) and H1299 (del-TP53/wt-RB1)), and normal human lung fibroblast (NHLF) lines (CCD-16 and MRC-9). Results of clonogenic assays indicated that Ad-mda7 enhanced the radiosensitivity of the NSCLC cells independent of their TP53 gene status. On the other hand, the NHLF cell lines seemed to be relatively resistant to the cytotoxic effects of Ad-mda7 and were not radiosensitized compared with the NSCLC cells. We further examined the basis for this difference in the ability of Ad-mda7 to radiosensitize NSCLC cells compared with normal cells. Radiation-induced apoptosis was restored in the NSCLC lines, but not in the normal lines. Western blot analysis revealed that Ad-mda7 enhances radiosensitivity independently of any ability to upregulate the expression of Fas or Bax in NSCLC cells. Further analysis indicated that phosphorylated c-Jun expression was increased by Ad-mda7 in both A549 and H1299 cells, but not in CCD-16 cells. These results support the use of gene replacement with Ad-mda7 in combination with radiotherapy for the treatment of NSCLC.

Adenoviridae↗